Validation of Salamander Dermal Mucus Swabs as a Novel, Nonlethal Approach for Amphibian Metabolomics and Glutathione Analysis Following Pesticide Exposure.

Evaluating biomarkers of stress in amphibians is critical to conservation, yet current techniques are often destructive and/or time-consuming, which limits ease of use. In the present study, we validate the use of dermal swabs in spotted salamanders (Ambystoma maculatum) for biochemical profiling, as well as glutathione (GSH) stress response following pesticide exposure. Thirty-three purchased spotted salamanders were acclimated to laboratory conditions at Washington College (Chestertown, MD, USA) for 4 weeks. Following acclimation, salamanders were randomly sorted into three groups for an 8-h pesticide exposure on soil: control with no pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), or chlorpyrifos. Before and after exposure, mucus samples were obtained by gently rubbing a polyester-tipped swab 50 times across the ventral and dorsal surfaces. Salamanders were humanely euthanized and dissected to remove the brain for acetylcholinesterase and liver for GSH and hepatic metabolome analyses, and a whole-body tissue homogenate was used for pesticide quantification. Levels of GSH were present in lower quantities on dermal swabs relative to liver tissues for chlorpyrifos, 2,4-D, and control treatments. However, 2,4-D exposures demonstrated a large effect size increase for GSH levels in livers (Cohen's d = 0.925, p = 0.036). Other GSH increases were statistically insignificant, and effect sizes were characterized as small for 2,4-D mucosal swabs (d = 0.36), medium for chlorpyrifos mucosal swabs (d = 0.713), and negligible for chlorpyrifos liver levels (d = 0.012). The metabolomics analyses indicated that the urea cycle, alanine, and glutamate metabolism biological pathways were perturbed by both sets of pesticide exposures. Obtaining mucus samples through dermal swabbing in amphibians is a viable technique for evaluating health in these imperiled taxa. Environ Toxicol Chem 2024;43:1126-1137. © 2024 SETAC.

Vibrio alginolyticus growth kinetics and the metabolic effects of iron.

IMPORTANCE: Transmission of V. alginolyticus occurs opportunistically through direct seawater exposure and is a function of its abundance in the environment. Like other Vibrio spp., V. alginolyticus are considered conditionally rare taxa in marine waters, with populations capable of forming large, short-lived blooms under specific environmental conditions, which remain poorly defined. Prior research has established the importance of temperature and salinity as the major determinants of Vibrio geographical and temporal range. However, bloom formation can be strongly influenced by other factors that may be more episodic and localized, such as changes in iron availability. Here we confirm the broad temperature and salinity tolerance of V. alginolyticus and demonstrate the importance of iron supplementation as a key factor for growth in the absence of thermal or osmotic stress. The results of this research highlight the importance of episodic iron input as a crucial metric to consider for the assessment of V. alginolyticus risk.

Distribution of Antibiotic Resistance in a Mixed-Use Watershed and the Impact of Wastewater Treatment Plants on Antibiotic Resistance in Surface Water.

The aquatic environment has been recognized as a source of antibiotic resistance (AR) that factors into the One Health approach to combat AR. To provide much needed data on AR in the environment, a comprehensive survey of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotic residues was conducted in a mixed-use watershed and wastewater treatment plants (WWTPs) within the watershed to evaluate these contaminants in surface water. A culture-based approach was used to determine prevalence and diversity of ARB in surface water. Low levels of AR Salmonella (9.6%) and Escherichia coli (6.5%) were detected, while all Enterococcus were resistant to at least one tested antibiotic. Fewer than 20% of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (17.3%) and carbapenem-resistant Enterobacteriaceae (CRE) (7.7%) were recovered. Six ARGs were detected using qPCR, primarily the erythromycin-resistance gene, ermB. Of the 26 antibiotics measured, almost all water samples (98.7%) had detectable levels of antibiotics. Analysis of wastewater samples from three WWTPs showed that WWTPs did not completely remove AR contaminants. ARGs and antibiotics were detected in all the WWTP effluent discharges, indicating that WWTPs are the source of AR contaminants in receiving water. However, no significant difference in ARGs and antibiotics between the upstream and downstream water suggests that there are other sources of AR contamination. The widespread occurrence and abundance of medically important antibiotics, bacteria resistant to antibiotics used for human and veterinary purposes, and the genes associated with resistance to these antibiotics, may potentially pose risks to the local populations exposed to these water sources.

Estimating dermal contact soil exposure for amphibians.

Chemical exposure estimation through the dermal route is an underemphasized area of ecological risk assessment for terrestrial animals. Currently, there are efforts to create exposure models to estimate doses from this pathway for use in ecological risk assessment. One significant limitation has been insufficient published data to characterize exposure and to support the selection and parameterization of appropriate models, particularly for amphibians in terrestrial habitats. Recent publications measuring pesticide doses to terrestrial-phase amphibians have begun to rectify this situation. We collated and summarized available measurements of terrestrial amphibian dermal exposure to pesticides from 11 studies in which researchers measured tissue concentrations associated with known pesticide experimental application rates. This data set included tissue concentrations in 11 amphibian species and 14 different pesticides. We then compared the results of two screening exposure models that differed based on surface area scaling approaches as a function of body weight (one based on birds as surrogates for amphibians and another amphibian-specific) to the measured tissue residue concentrations. We define a false-negative rate for each screening model as the proportion of amphibians for which the predicted concentration is less than the observed concentration (i.e., underestimate), contrary to the intent of screening models, which are intended to have a bias for higher exposure concentrations. The screening model that uses birds as surrogates did not have any instances where estimated expected avian doses were less than measured amphibian body burdens. When using the amphibian-specific exposure model that corrected for differences between avian and amphibian surface area, measured concentrations were greater than model estimates for 11.3% of the 1158 comparisons. The database of measured pesticide concentrations in terrestrial amphibians is provided for use in calculating bioconcentration factors and for future amphibian dermal exposure model development. Integr Environ Assess Manag 2023;19:9-16. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Differentiating metabolomic responses of amphibians to multiple stressors.

One of the biggest challenges in ecological risk assessment is determining the impact of multiple stressors on individual organisms and populations in real world scenarios. Frequently, data derived from laboratory studies of single stressors are used to estimate risk parameters and do not adequately address scenarios where other stressors exist. Emerging 'omic technologies, notably metabolomics, provide an opportunity to address the uncertainties surrounding ecological risk assessment of multiple stressors. The objective of this study was to use metabolomic profiling to investigate the effect of multiple stressors on amphibian metamorphs. We exposed post-metamorphosis (180 days) southern leopard frogs (Lithobates sphenocephala) to the insecticide carbaryl (480 µg/L), predation stress, and a combined pesticide and predation stress treatment. Corticosterone analysis revealed mild support for an induction in response to predation stress alone but strongly suggests that carbaryl exposure, alone or in combination with predation cues, can significantly elevate this known biomarker in amphibians. Metabolomics analysis accurately classed, based on relative nearness, carbaryl and predation induced changes in the hepatic metabolome and biochemical fluxes appear to be associated with a similar biological response. Support vector machine analysis with recursive feature elimination of the acquired metabolomic spectra demonstrated 85-96% classification accuracy among control and all treatment groups when using the top 75 ranked retention time bins. Biochemical fluxes observed in the groups exposed to carbaryl, predation, and the combined treatment include amino acids, sugar derivatives, and purine nucleotides. Ultimately, this methodology could be used to interpret short-term toxicity assays and the presence of environmental stressors to overall metabolomic effects in non-target organisms.

Using metabolomic profiling to inform use of surrogate species in ecological risk assessment practices.

The U.S. EPA frequently uses avian or fish toxicity data to set protective standards for amphibians in ecological risk assessments. However, this approach does not always adequately represent aquatic-dwelling and terrestrial-phase amphibian exposure data. For instance, it is accepted that early life stage tests for fish are typically sensitive enough to protect larval amphibians, however, metamorphosis from tadpole to a terrestrial-phase adult relies on endocrine cues that are less prevalent in fish but essential for amphibian life stage transitions. These differences suggest that more robust approaches are needed to adequately elucidate the impacts of pesticide exposure in amphibians across critical life stages. Therefore, in the current study, methodology is presented that can be applied to link the perturbations in the metabolomic response of larval zebrafish (Danio rerio), a surrogate species frequently used in ecotoxicological studies, to those of African clawed frog (Xenopus laevis) tadpoles following exposure to three high-use pesticides, bifenthrin, chlorothalonil, or trifluralin. Generally, D. rerio exhibited greater metabolic perturbations in both number and magnitude across the pesticide exposures as opposed to X. laevis. This suggests that screening ecological risk assessment surrogate toxicity data would sufficiently protect amphibians at the single life stage studied but care needs to be taken to understand the suite of metabolic requirements of each developing species. Ultimately, methodology presented, and data gathered herein will help inform the applicability of metabolomic profiling in establishing the risk pesticide exposure poses to amphibians and potentially other non-target species.

Induced Hepatic Glutathione and Metabolomic Alterations Following Mixed Pesticide and Fertilizer Exposures in Juvenile Leopard Frogs (Lithobates sphenocephala).

The increasing use of agrochemicals, alone and in combination, has been implicated as a potential causative factor in the decline of amphibians worldwide. Fertilizers and pesticides are frequently combined into single-use tank mixtures for agricultural applications to decrease costs while meeting the food demands of a growing human population. Limited data are available on the effects of increased nitrogen levels in nontarget species, such as amphibians, and therefore investigating alterations in the nitrogen cycle and its impacts on amphibians needs to be considered in best management practices going forward. The objective of the present study was to elucidate the impact of fertilizer (urea) and herbicide (atrazine and/or alachlor) tank mixtures on the hepatic metabolome of juvenile leopard frogs as well as to investigate alterations in oxidative stress by relating these changes to glutathione (GSH) levels. Herbicide exposure only moderately increased this parameter in amphibians, however, urea alone and in combination with either atrazine or alachlor statistically elevated GSH levels. Interestingly, urea also inhibited pesticide uptake: calculated bioconcentration factors were greatly decreased for atrazine and alachlor when urea was present in the exposure mixture. Metabolomic profiling identified fluxes in hepatic metabolites that are involved in GSH and carbohydrate metabolic processes as well as altered intermediates in the urea cycle. Ultimately, understanding the biological impacts of nitrogenous fertilizers alone and in combination with pesticide exposure will inform best management practices to conserve declining amphibian populations worldwide. Environ Toxicol Chem 2022;41:122-133. © 2021 SETAC.

Route of exposure influences pesticide body burden and the hepatic metabolome in post-metamorphic leopard frogs.

Pesticides are being applied at a greater extent than in the past. Once pesticides enter the ecosystem, many environmental factors can influence their residence time. These interactions can result in processes such as translocation, environmental degradation, and metabolic activation facilitating exposure to target and non-target species. Most anurans start off their life cycle in aquatic environments and then transition into terrestrial habitats. Their time in the aquatic environment is generally short; however, many important developmental stages occur during this tenure. Post-metamorphosis, most species spend many years on land but migrate back to the aquatic environment for breeding. Due to the importance of both the aquatic and terrestrial environments to the life stages of amphibians, we investigated how the route of exposure (i.e., uptake from contaminated soils vs. uptake from contaminated surface water) influences pesticide bioavailability and body burden for four pesticides (bifenthrin (BIF), chlorpyrifos (CPF), glyphosate (GLY), and trifloxystrobin (TFS)) as well as the impact on the hepatic metabolome of adult leopard frogs (Gosner stage 46 with 60-90 days post-metamorphosis). Body burden concentrations for amphibians exposed in water were significantly higher (ANOVA p < 0.0001) compared to amphibians exposed to contaminated soil across all pesticides studied. Out of 80 metabolites that were putatively identified, the majority expressed a higher abundance in amphibians that were exposed in pesticide contaminated water compared to soil. Ultimately, this research will help fill regulatory data gaps, aid in the creation of more accurate amphibian dermal uptake models and inform continued ecological risk assessment efforts.

Honey Bees and Neonicotinoid-Treated Corn Seed: Contamination, Exposure, and Effects.

Most corn (Zea mays) seeds planted in the United States in recent years are coated with a seed treatment containing neonicotinoid insecticides. Abrasion of the seed coating generates insecticide-laden planter dust that disperses through the landscape during corn planting and has resulted in many "bee-kill" incidents in North America and Europe. We investigated the linkage between corn planting and honey bee colony success in a region dominated by corn agriculture. Over 3 yr we consistently observed an increased presence of corn seed treatment insecticides in bee-collected pollen and elevated worker bee mortality during corn planting. Residues of seed treatment neonicotinoids, clothianidin and thiamethoxam, detected in pollen positively correlated with cornfield area surrounding the apiaries. Elevated worker mortality was also observed in experimental colonies fed field-collected pollen containing known concentrations of corn seed treatment insecticides. We monitored colony growth throughout the subsequent year in 2015 and found that colonies exposed to higher insecticide concentrations exhibited slower population growth during the month of corn planting but demonstrated more rapid growth in the month following, though this difference may be related to forage availability. Exposure to seed treatment neonicotinoids during corn planting has clear short-term detrimental effects on honey bee colonies and may affect the viability of beekeeping operations that are dependent on maximizing colony size in the springtime. Environ Toxicol Chem 2021;40:1212-1221. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Cross-Taxa Distinctions in Mechanisms of Developmental Effects for Aquatic Species Exposed to Trifluralin.

Standard ecological risk assessment practices often rely on larval and juvenile fish toxicity data as representative of the amphibian aquatic phase. Empirical evidence suggests that endpoints measured in fish early life stage tests are often sufficient to protect larval amphibians. However, the process of amphibian metamorphosis relies on endocrine cues that affect development and morphological restructuring and are not represented by these test endpoints. The present study compares developmental endpoints for zebrafish (Danio rerio) and the African clawed frog (Xenopus laevis), 2 standard test species, exposed to the herbicide trifluralin throughout the larval period. Danio rerio were more sensitive and demonstrated a reduction in growth measurements with increasing trifluralin exposure. Size of X. laevis at metamorphosis was not correlated with exposure concentration; however, time to metamorphosis was delayed relative to trifluralin concentration. Gene expression patterns indicate discrepancies in response by D. rerio and X. laevis, and dose-dependent metabolic activity suggests that trifluralin exposure perturbed biological pathways differently within the 2 species. Although many metabolites were correlated with exposure concentration in D. rerio, nontargeted hepatic metabolomics identified a subset of metabolites that exhibited a nonmonotonic response to trifluralin exposure in X. laevis. Linking taxonomic distinctions in cellular-level response with ecologically relevant endpoints will refine assumptions used in interspecies extrapolation of standard test effects and improve assessment of sublethal impacts on amphibian populations. Environ Toxicol Chem 2020;39:1797-1812. Published 2020. This article is a US government work and is in the public domain in the USA.

Agrochemical Mixtures and Amphibians: The Combined Effects of Pesticides and Fertilizer on Stress, Acetylcholinesterase Activity, and Bioaccumulation in a Terrestrial Environment.

Tank mixtures are popular within the agricultural community because they are time- and cost-effective, but field applications leave nontarget organisms at risk of exposure. We explored the effects of a common herbicide (atrazine and alachlor) and fertilizer (urea) tank mixture on juvenile frog corticosterone stress levels, acetylcholinesterase (AChE) activity, and pesticide bioaccumulation. Single agrochemical or tank mixtures were applied to terrestrial microcosms, and then individual Southern leopard frog (Lithobates sphenocephala) juveniles were added to microcosms for an 8-h exposure. Afterward, frogs were transferred to aquatic microcosms for 1 h to monitor corticosterone prior to euthanasia, brain tissues were excised to evaluate AChE, and tissue homogenates were analyzed for pesticide bioconcentation with gas chromatography-mass spectrometry. Atrazine significantly increased corticosterone in frogs, particularly when combined with alachlor and urea. Atrazine increased AChE and urea decreased AChE, although no interactive effects of chemical combinations were discernible. Relative to their individual treatments, the complete tank mixture with all 3 agrochemicals resulted in 64% greater bioconcentration of atrazine and 54% greater bioconcentration of alachlor in frog tissues. Our results suggest that agrochemical mixtures as well as their active ingredients can lead to altered stress levels and impaired physiological responses in amphibians. An improved understanding of the effects of co-exposure to environmental contaminants in amphibians is important in assessing the ecological risks these compounds pose. Environ Toxicol Chem 2019;9999:1-10. © 2019 SETAC.

Influence of exposure to pesticide mixtures on the metabolomic profile in post-metamorphic green frogs (Lithobates clamitans).

Pesticide use in agricultural areas requires the application of numerous chemicals to control target organisms, leaving non-target organisms at risk. The present study evaluates the hepatic metabolomic profile of one group of non-target organisms, amphibians, after exposure to a single pesticide and pesticide mixtures. Five common-use pesticide active ingredients were used in this study, three herbicides (atrazine, metolachlor and 2,4-d), one insecticide (malathion) and one fungicide (propiconazole). Juvenile green frogs (Lithobates clamitans) were reared for 60-90days post-metamorphosis then exposed to a single pesticide or a combination of pesticides at the labeled application rate on soil. Amphibian livers were excised for metabolomic analysis and pesticides were quantified for whole body homogenates. Based on the current study, metabolomic profiling of livers support both individual and interactive effects where pesticide exposures altered biochemical processes, potentially indicating a different response between active ingredients in pesticide mixtures, among these non-target species. Amphibian metabolomic response is likely dependent on the pesticides present in each mixture and their ability to perturb biochemical networks, thereby confounding efforts with risk assessment.

Analysis of pesticides in surface water, stemflow, and throughfall in an agricultural area in South Georgia, USA.

To study spray drift contributions to non-targeted habitats, pesticide concentrations in stemflow (water flowing down the trunk of a tree during a rain event), throughfall (water from tree canopy only), and surface water in an agriculturally impacted wetland area near Tifton, Georgia, USA were measured (2015-2016). Agricultural fields and sampling locations were on the University of Georgia's Gibbs Research Farm, Tifton, GA. Samples were screened for more than 160 pesticides, and cumulatively, 32 different pesticides were detected across matrices. Data indicate that herbicides and fungicides were present in all types of environmental samples analyzed while insecticides were only detected in surface water samples. The highest pesticide concentration observed was 10.50 µg/L of metolachlor in an August 2015 surface water sample. Metolachlor, tebuconazole, and fipronil were the most frequently detected herbicide, fungicide, and insecticide, respectively, regardless of sample origin. The most frequently detected pesticide in surface water and stemflow samples was metolachlor (0.09-10.5 µg/L), however, the most commonly detected pesticide in throughfall samples was biphenyl (0.02-0.07 µg/L). These data help determine the importance of indirect chemical exposures to non-targeted habitats by assessing inputs from stemflow and throughfall into surface waters.

Effect of hydration status on pesticide uptake in anurans following exposure to contaminated soils.

In this study, the impact of hydration status on dermal uptake of pesticides in two species of amphibians is examined. Absorption of pesticides in anurans occurs primarily through a highly vascularized dermal seat patch; however, pesticides can also enter through the superficial dermis following exposure. Despite the growing body of literature on dermal exposure in amphibians, little is known on how hydration status influences uptake. Thus, the objective of this study was to investigate the influence of hydration status on absorption of pesticides (atrazine, triadimefon, metolachlor, chlorothalonil, and imidacloprid) in southern leopard frogs (Lithobates sphenocephala) and Fowler's toads (Anaxyrus fowleri). Amphibian treatments included dehydration periods of 0, 2, 4, 6, 8, or 10 h prior to exposure to pesticide-contaminated soils for 8 h. Following exposure, soil and whole-body homogenates were extracted and analyzed by LC-MS/MS. Dehydration time was then regressed against post-exposure concentrations to infer the impact of dehydration on dermal pesticide uptake. Increased dehydration time resulted in significantly lowered pesticide concentrations in both species (F6, 293 = 67.66, p = 0.007) for the five pesticides studied. This phenomenon could be due to an energy and/or dilution effect.

Using in vitro derived enzymatic reaction rates of metabolism to inform pesticide body burdens in amphibians.

Understanding how pesticide exposure to non-target species influences toxicity is necessary to accurately assess the ecological risks these compounds pose. To assess the potential metabolic activation of broad use pesticides in amphibians, in vitro and in vivo metabolic rate constants were derived from toad (Anaxyrus terrestris) livers in experiments measuring the depletion of atrazine (ATZ), triadimefon (TDN), and fipronil (FIP) as well as formation of their metabolites. To determine the predictability of these in vitro derived rate constants, Fowler's toads (Anaxyrus fowleri) were exposed to soil contaminated with each of the pesticides at maximum application rate. Desethyl atrazine (DEA) and deisopropyl atrazine (DIA), both metabolites of ATZ, exhibited similar velocities (Vmax) while the KM constant for DIA was two times higher than DEA. TDN was metabolized into two diastereomers of triadimenol (TDL A and TDL B), where TDL B had a Vmax around two times higher than TDL A. The metabolite fipronil sulfone's Vmax and KM were 150 pmol min-1 mg-1 and 29 µM, respectively. While intrinsic clearance rates for the pesticides ranged from 0.54 to 38.31 mL min-1 kg-1. Thus, gaining knowledge on differences in metabolism of pesticides within amphibians is important in estimating risk to these non-target species since the inherent toxicity of metabolites can differ from the parent compound.

Endogenous and exogenous biomarker analysis in terrestrial phase amphibians (Lithobates sphenocephala) following dermal exposure to pesticide mixtures.

Pesticide mixtures are frequently co-applied throughout an agricultural growing season to maximize crop yield. Therefore, non-target ecological species (e.g., amphibians) may be exposed to several pesticides at any given time on these agricultural landscapes. The objectives of this study were to quantify body burdens in terrestrial phase amphibians and translate perturbed metabolites to their corresponding biochemical pathways affected by exposure to pesticides as both singlets and in combination. Southern leopard frogs (Lithobates sphenocephala) were exposed either at maximum or 1/10th maximum application rate to single, double, or triple pesticide mixtures of bifenthrin (insecticide), metolachlor (herbicide), and triadimefon (fungicide). Tissue concentrations demonstrate both facilitated and competitive uptake of pesticides when in mixtures. Metabolomic profiling of amphibian livers identified metabolites of interest for both application rates, however; magnitude of changes varied for the two exposure rates. Exposure to lower concentrations demonstrated down regulation in amino acids, potentially due to their being utilized for glutathione metabolism and/or increased energy demands. Amphibians exposed to the maximum application rate resulted in up regulation of amino acids and other key metabolites likely due to depleted energy resources. Coupling endogenous and exogenous biomarkers of pesticide exposure can be utilized to form vital links in an ecological risk assessment by relating internal dose to pathophysiological outcomes in non-target species.

Biomarker analysis of American toad (Anaxyrus americanus) and grey tree frog (Hyla versicolor) tadpoles following exposure to atrazine.

The objective of the current study was to use a biomarker-based approach to investigate the influence of atrazine exposure on American toad (Anaxyrus americanus) and grey tree frog (Hyla versicolor) tadpoles. Atrazine is one of the most frequently detected herbicides in environmental matrices throughout the United States. In surface waters, it has been found at concentrations from 0.04-2859µg/L and thus presents a likely exposure scenario for non-target species such as amphibians. Studies have examined the effect of atrazine on the metamorphic parameters of amphibians, however, the data are often contradictory. Gosner stage 22-24 tadpoles were exposed to 0 (control), 10, 50, 250 or 1250µg/L of atrazine for 48h. Endogenous polar metabolites were extracted and analyzed using gas chromatography coupled with mass spectrometry. Statistical analyses of the acquired spectra with machine learning classification models demonstrated identifiable changes in the metabolomic profiles between exposed and control tadpoles. Support vector machine models with recursive feature elimination created a more efficient, non-parametric data analysis and increased interpretability of metabolomic profiles. Biochemical fluxes observed in the exposed groups of both A. americanus and H. versicolor displayed perturbations in a number of classes of biological macromolecules including fatty acids, amino acids, purine nucleosides, pyrimidines, and mono- and di-saccharides. Metabolomic pathway analyses are consistent with findings of other studies demonstrating disruption of amino acid and energy metabolism from atrazine exposure to non-target species.

Effects of triclosan on bacterial community composition and Vibrio populations in natural seawater microcosms.

Pharmaceuticals and personal care products, including antimicrobials, can be found at trace levels in treated wastewater effluent. Impacts of chemical contaminants on coastal aquatic microbial community structure and pathogen abundance are unknown despite the potential for selection through antimicrobial resistance. In particular, Vibrio, a marine bacterial genus that includes several human pathogens, displays resistance to the ubiquitous antimicrobial compound triclosan. Here we demonstrated through use of natural seawater microcosms that triclosan (at a concentration of ~5 ppm) can induce a significant Vibrio growth response (68-1,700 fold increases) in comparison with no treatment controls for three distinct coastal ecosystems: Looe Key Reef (Florida Keys National Marine Sanctuary), Doctors Arm Canal (Big Pine Key, FL), and Clam Bank Landing (North Inlet Estuary, Georgetown, SC). Additionally, microbial community analysis by 16 S rRNA gene sequencing for Looe Key Reef showed distinct changes in microbial community structure with exposure to 5 ppm triclosan, with increases observed in the relative abundance of Vibrionaceae (17-fold), Pseudoalteromonadaceae (65-fold), Alteromonadaceae (108-fold), Colwelliaceae (430-fold), and Oceanospirillaceae (1,494-fold). While the triclosan doses tested were above concentrations typically observed in coastal surface waters, results identify bacterial families that are potentially resistant to triclosan and/or adapted to use triclosan as a carbon source. The results further suggest the potential for selection of Vibrio in coastal environments, especially sediments, where triclosan may accumulate at high levels.

Soil organic matter content effects on dermal pesticide bioconcentration in American toads (Bufo americanus).

Pesticides have been implicated as a major factor in global amphibian declines and may pose great risk to terrestrial phase amphibians moving to and from breeding ponds on agricultural landscapes. Dermal uptake from soil is known to occur in amphibians, but predicting pesticide availability and bioconcentration across soil types is not well understood. The present study was designed to compare uptake of 5 current-use pesticides (imidacloprid, atrazine, triadimefon, fipronil, and pendimethalin) in American toads (Bufo americanus) from exposure on soils with significant organic matter content differences (14.1% = high organic matter and 3.1% = low organic matter). We placed toads on high- or low-organic matter soil after applying individual current-use pesticides on the soil surface for an 8-h exposure duration. Whole body tissue homogenates and soils were extracted and analyzed using liquid chromatography-mass spectrometry to determine pesticide tissue and soil concentration, as well as bioconcentration factor in toads. Tissue concentrations were greater on the low-organic matter soil than the high-organic matter soil across all pesticides (average ± standard error; 1.23 ± 0.35 ppm and 0.78 ± 0.23 ppm, respectively), and bioconcentration was significantly higher for toads on the low-organic matter soil (analysis of covariance p = 0.002). Soil organic matter is known to play a significant role in the mobility of pesticides and bioavailability to living organisms. Agricultural soils typically have relatively lower organic matter content and serve as a functional habitat for amphibians. The potential for pesticide accumulation in amphibians moving throughout agricultural landscapes may be greater and should be considered in conservation and policy efforts. Environ Toxicol Chem 2016;35:2734-2741. © 2016 SETAC.

Pesticide Uptake Across the Amphibian Dermis Through Soil and Overspray Exposures.

For terrestrial amphibians, accumulation of pesticides through dermal contact is a primary route of exposure in agricultural landscapes and may be contributing to widespread amphibian declines. To show pesticide transfer across the amphibian dermis at permitted label application rates, our study was designed to measure pesticide body burdens after two simulated exposure scenarios. We compared direct exposures, where amphibians were present when spraying occurred, to indirect exposures, where amphibians were exposed to soils after pesticide application. During summer 2012, we reared barking (Hyla gratiosa) and green treefrogs (H. cinerea) through 60-90 days post-metamorphosis at a United States Environmental Protection Agency research laboratory. We tested exposure for 8 h to five pesticide active ingredients (imidacloprid, atrazine, triadimefon, fipronil, or pendimethalin) in glass aquaria lined with soil in the laboratory. We quantified total pesticide body burden and soil concentrations using liquid chromatography-mass spectrometry. All individuals in both treatments had measurable body burdens at the end of the study. A randomized block design analysis of variance (n = 18) showed that body burdens (p = 0.03) and bioconcentration factors (BCFs) (p = 0.01) were significantly greater in the direct overspray treatment relative to the indirect soil spray treatment for both species and tested pesticides. BCFs ranged from 0.1 to 1.16 and from 0.013 to 0.78 in the direct and indirect treatments, respectively. Our study shows dermal uptake for multiple pesticides from both direct spray and indirect soil exposures and provides empirical support for the degree to which terrestrial phase amphibians have higher body burdens after overspray pesticide exposure.